Crystal Structure of Mesaconyl-CoA Hydratase from Methylorubrum extorquens CM4

Methylorubrum extorquens, a facultative methylotroph, assimilates C1 compounds and accumulates poly-β-hydroxylbutyrate (PHB) as carbon and energy sources. The ethylmalonyl pathway is central to the carbon metabolism of M. extorquens, and is linked with a serine cycle and a PHB biosynthesis pathway. Understanding the ethylmalonyl pathway is vital in utilizing methylotrophs to produce value-added chemicals. In this study, we determined the crystal structure of the mesaconyl-CoA hydratase from M. extorquens (MeMeaC) that catalyzes the reversible conversion of mesaconyl-CoA to β-methylmalyl-CoA. The crystal structure of MeMeaC revealed that the enzyme belongs to the MaoC-like dehydratase domain superfamily and functions as a trimer. In our current MeMeaC structure, malic acid occupied the substrate binding site, which reveals how MeMeaC recognizes the β-methylmalyl-moiety of its substrate. The active site of the enzyme was further speculated by comparing its structure with those of other MaoC-like hydratases.

In spite of the progress in the study of MeaC, there have been no reports on the structure containing the substrate or its analogues in the active site so far. It is not easy to present the substrate specificity at the molecular level or reveal the molecular mechanism without a structure. Thus, in this study, we determined the crystal structure of MeaC from Methylorubrum extorquens CM4 (MeMeaC). From the structural information for the MeMeaC structure containing the analogue, we suggest the molecular function and mechanism, which will contribute to understanding ethylmalonyl-CoA pathway as well as the 3-hydroxypropionate cycle for autotrophic CO 2 fixation.

Expression and Purification
MeMeaC (Mchl_4075) was amplified with forward and reverse primers designed as 5'-GCGCCATATGAA GACCAATCCGGGCCGCTTCTTC-3' and 5'-ATATCTCGAGGCGCGGGATGAAGGCCCAATAGTC-3' , respectively. The amplified gene was cloned into pET30a expression vector using NdeI and XhoI restriction enzymes. The pET30a:MeMeaC was transformed into the E. coli strain BL21(DE3)-T1 R . Cells were cultured in an LB medium containing kanamycin at 37ºC, until reaching an absorbance of 0.7 at 600 nm. After induction using 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), cells were incubated for 18 h at 20ºC. Following harvest, the cell pellet was resuspended in A buffer (40 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5 mM βmercaptoethanol) and disrupted by ultrasonication with a pulse. The cell debris was removed by centrifugation at 13,000 ×g for 1 h. The lysate was bound to Ni-NTA agarose (Qiagen, Germany) by gravity. After washing with lysis buffer containing 30 mM imidazole, the protein was eluted by using B buffer (40 mM Tris-HCl, pH 8.0, 150 mM NaCl, 300 mM Imidazole, and 5 mM β-mercaptoethanol). To improve purity for crystallization, the protein was further purified by size exclusion chromatography using a HiPrep 2.6/60 Sephacryl S-300 HR column (Cytiva, USA). The purified protein was concentrated to 68 mg/ml in a solution with the buffer A.

Crystallization
Initial crystallization of the purified protein was carried out by using commercial screening solutions, including Index, PEG Ion Screen I and II (Hampton Research, USA), and Wizard Classic I and II (Rigaku Reagents, USA), as the sitting-drop vapor-diffusion method at 20°C. We mixed 1.0 μl of the protein solution with 1.0 μl of the reservoir solution and then equilibrated this against 500 μl of the reservoir solution. Then, we identified crystals at conditions of 0.2 M DL-malic acid (pH 7.0) or 0.2 M sodium malonate (pH 7.0) with 20% (w/v) PEG3350 precipitant. After adjusting protein concentration, we could obtain suitable crystals under 0.2 M DL-malic acid (pH 7.0) and 20% (w/v) PEG3350 in 1-3 days.

Data Collection and Processing
A cryo-protectant containing 30% (v/v) glycerol in a reservoir solution was used for X-ray diffraction. The datasets of the native protein were collected at 100 K at the 7A beamline of the Pohang Accelerator Laboratory (PAL, Republic of Korea), using a Quantum 270 CCD detector (ADSC, USA). The best crystal diffracted to 1.95 Å resolution. The collected data was commonly indexed, integrated, and scaled using the HKL2000 suite (HKL Research, USA). The crystal belonged to space group P6 3 with the following unit cell parameters: a = 79.224 Å, b = 79.224 Å, c = 91.008 Å, α = β = 90°, and γ = 120°. The data statistics are summarized in Table 1.

Structure Determination and Refinement
The MeaC structure was determined by the molecular replacement method with the CCP4 version of MOLREP [17]. The crystal structure of the putative MaoC domain protein dehydratase from Chloroflexus aurantiacus J-10fl (PDB code 4E3E) was used as a search model. The initial model building was automatically performed using ARP/wARP, and the final model was built by using the program WinCoot [18,19]. The refinement was performed with REFMAC5 in CCP4 suite [20,21]. The refined model was deposited in the Protein Data Bank with the PDB code 8HGN. Data collection and refinement statistics are given in Table 1.

Overall Structure of MeMeaC
To elucidate the molecular mechanism and substrate binding mode of MeMeaC, we determined the crystal structure of the enzyme at a 1.95 Å resolution with R work = 20.9% and R free = 27.8%, respectively. The crystal structure of MeMeaC revealed that the enzyme belongs to the MaoC-like dehydratase domain superfamily, which consists of two central helices and a curved eleven-stranded antiparallel β-sheet (Fig. 2B). MeMeaC is composed of an N-terminal subdomain (MeaC N , residue 1~147), a C-terminal subdomain (MeaC C , residue 191~348), and a bridge (MeaC Br , residue 148~190) linking the two subdomains. Each subdomain with a hotdog topology is built of a curved, five-stranded antiparallel β-sheet (MeaC N : β1-β4-β5-β6-β3 and MeaC C : β7-β10-β11-β12-β9) that wraps around the central helix (MeaC N : α4 and MeaC C : α7) [22]. The overall structure of MeMeaC is maintained by the interaction between MeaC N and MeaC C with extensive non-covalent contacts, including a long antiparallel βsheet formed from β3-β9 (Fig. 2B). MeaCBr connects MeaC N to MeaC C by crossing over the saddle side generated by their interactions. When we compared MeMeaC with MaoC-like hydratase from Phytophthora capsici (PDB code 3KH8) and 2-enoyl-CoA hydratase from Candida tropicalis (PDB code 1PN4), which are divided into three parts (MeaC N , MeaC C , and MeaC Br ), we found that the connection by MeaC Br differ from others (Fig. 2C). While other bridges head directly to the end of central β-sheet of a C-terminal subdomain, MeaC Br passes over between α3 of MeaC N and the loop of β10-β11 in MeaC C . Particularly, residue 177 ~190 on MeaC Br is grasped by Arg194, Asn266, Asg303, Asp305 on MeaC N , and Thr43, Ile54, Phe50 on MeaC C (Fig. 2D). This grip enhances the MeaC N -MeaC C interaction, which could contribute to maintaining the overall scaffold of MeMeaC.
Although one molecule is present in the asymmetric unit of the crystal structure, the P6 3 crystallographic symmetry operation of MeMeaC revealed that the enzyme forms a trimer. PDBePISA (Proteins, Interfaces, Structures, and Assemblies) also suggested that MeMeaC forms a trimer [23]. The trimer is assembled by the interaction between interfaces A and B with buried surface areas of 1548.8 Å and 1505.3 Å, respectively (Figs. 3A-3C). Taking account of the wide area of interface A and B in the trimer assembly, we speculated that MeMeaC could stably maintain the trimer in solution. When the size-exclusive chromatography experiment was performed, the molecular weight of MeMeaC was calculated as 105.3 kDa, similar to a trimer, which confirms the trimerization in solution (Fig. 3D).

CoA Binding Site of MeMeaC
To elucidate the substrate binding mode of MeMeaC, we attempted to determine the complex structure with the CoA molecule, however, both soaking and co-crystallization experiments failed. Alternatively, superposition of the structure of MeMeaC with those of enoyl-CoA hydratases in complex with CoA (PDB codes: 1PN4 and 4WNB) revealed that the 6xHis-tag of MeMeaC is positioned in the vicinity of the CoA binding site and interferes with the binding of CoA into the enzyme (Figs. 4A and 4B). Among structures of MaoC-like enoyl-CoA hydratases, 2-enoyl-CoA hydratase from C. tropicalis (PDB code 1PN4) and ChsH1-ChsH2 complex in Mycobacterium tuberculosis (PDB code 4WNB) contain (3R)-hydroxydecanoyl-CoA and 3-oxo-4-pregnene-20carboxyl-CoA (3-OPC-CoA), respectively [24,25]. Superposition of the MeMeaC structure with 1PN4 (RMSD = 5.9 Å) and 4WNB (RMSD = 7.7 Å) allowed us to identify the binding mode of CoA in MeMeaC. The CoA binding site might be formed between β4-loop-β5 and β10-loop-β11 on the saddle side formed by the combination of MeaC N and MeaC C , and particularly, the 3'-phosphoadenosine-moiety might be positioned near the β4-loop-β5 in MeMeaC (Fig. 4A). The crystal structures of MeMeaC, 2-enoyl-CoA hydratase from C. tropicalis (PDB code 1PN4) and ChsH1-ChsH2 complex in M. tuberculosis (PDB code 4WNB) show that conserved lysine is commonly located on β4-loop-β5 of their structure (Fig. 4A). The Lys103 of 1PN4 and Lys134 of 4WNB interact with the 3-phosphoadenosine-moiety of CoA. Thus, considering that Lys113 is also located at the corresponding position in MeMeaC ( Figs. 2A and 4A), these observations imply that Lys113 is a crucial residue for stabilization of the 3-phosphoadenosine-moiety of CoA near the β4-loop-β5 in MaoC-like enoyl-CoA hydratases, including MeMeaC.

Stabilization Mode of the β-Methylmalyl-Moiety
In our current structure of MeMeaC, we observed that a malic acid molecule, which is used as a main precipitant for the crystallization, is bound at the active site (Fig. 5A). When we superpose the structure of MeMeaC with 1PN4 and 4WNB, the malic acid molecule in MeMeaC was located at the position similar to (3R)hydroxydecanoyl-and 3-OPC-moiety bound in 1PN4 and 4WNB, respectively (Fig. 5B). Moreover, because the structure of malic acid is quite similar to the β-methylmalyl-and mesaconyl-moiety of β-methylmalyl-CoA and mesaconyl-CoA, respectively, we suspect that the β-methylmalyl-and mesaconyl-moiety might be stabilized in a mode similar to malic acid in the MeMeaC. The binding site of the malic acid molecule is formed at the interface between MeaC N and MeaC C (Fig. 5C). The conserved residues, such as Phe71, Ser79, Asn85, Arg142, Asn226, His231, Tyr248, and Gly249, were located at the malic acid binding site ( Figs. 2A and 5C). Based on the spatial position of these residues and the binding mode for malic acid, it could be inferred that they are required for holding the β-methylmalyl-and mesaconyl-moiety and performing the enzyme activity. The crystal structure of MeMeaC shows that these residues form polar contact and hydrophobic interaction for binding of the malic acid in the active site (Fig. 5C). Oxygen of Ser79 sidechain and nitrogen of Arg142 sidechain make hydrogen bonds to O1 and O2 of malic acid with 2.6 Å and 2.8 Å distances. Nitrogen atoms of Asn85 and Gly249 backbones form hydrogen bonds to O4 and O5 of malic acid with 2.6 Å and 3.0 Å distances. O3 of malic acid interacts with nitrogen atoms of sidechains of Asn226 and His231 as hydrogen bonds of 2.6 Å and 3.0 Å distances. There are hydrophobic interactions between Phe71, Tyr248, and the carbon backbone of the malic acid, which contribute to stabilizing binding β-methylmalyl-moiety at the active site.
Amongst these interactions, it is speculated that the double hydrogen bonds of Asn226-O3-His231 could play a pivotal role in the substrate recognition and the enzyme reaction. Taking into account that a β-methylmalyl-CoA has an additional hydroxyl group at the counterpart of O3 of the malic acid and is not at a mesaconyl-CoA, strong interaction of Asn226-O3-His231 could make the binding of a β-methylmalyl-moiety more stable, which contributes to improving the specificity. Moreover, when the MeMeaC structure is overlapped on MaoC-like hydratase from P. capsici (PDB code 3KH8, RMSD = 4.2 Å) and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (PDB code 1IQ6, RMSD = 3.9 Å) [26,27], Asn226 and His231, known as catalytic residues in other MaoC-Like hydratases, are also positioned in MeMeaC (Fig. 5D), suggesting that MeMeaC catalyzes the enzyme reaction a similar mode to other MaoC-like hydratases. Therefore, assuming that MeMeaC has the reaction mechanism via the concerted transition state like the proposed mechanism of 2-enoyl-CoA hydratase from C. tropicalis (PDB code 1PN4) [24], Asn226 and His231 might function as activating single water molecules for the hydration/dehydration reaction.
In summary, we determined the crystal structure of MeMeaC with a 1.95 Å resolution. Compared to the other structures of MaoC-like hydratases, the active pocket and CoA binding sites could be identified. The malic acid molecule bound in our structure assists in identifying the binding mode of the functional-moiety of the substrate. However, since it was hard to obtain the substrates, we were not able to measure in vitro activities for the mutant, which could solidify the molecular mechanism for the enzyme reaction and the substrate specificity. In the future, if the mutant activities are measured through in vivo and in vitro approaches, our structural analysis of MeMeaC will contribute to expanding the comprehension of the ethylmalonyl-CoA pathway and the 3-hydroxypropionate cycle by revealing the molecular mechanism for the reaction and substrate recognition of MeMeaC.
Methylotrophs such as M. extorquens utilize the ethylmalonyl-CoA pathway. M. extorquens is the best-studied methylotroph [8,[28][29][30] and is also the best platform for methanol-based biotechnology [31]. Considering that each intermediate in the ethylmalonyl-CoA pathway is a potential starting point for designing new pathways for producing high value-added chemicals [12], the understanding of meaC derived from this study may help in developing various material production methods using methanol based on M. extorquens in the future.  Overlapping putative catalytic residues. Putative catalytic residues are described as sticks. MaoClike hydratase from P. capsici (PDB code 3KH8) and (R)-specific enoyl coenzyme A hydratase from A. caviae (PDB code 1IQ6) are shown in light yellow and gold color, respectively.